High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca sativa)

Abstract

CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4), a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4, were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T1) and 368 T2 plants by deep amplicon sequencing revealed that 57% of T1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T1 and more than 100 T2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature.

CRISPR for Crop Improvement: An Update Review

“…Targeting NCED4 in a co-editing strategy could, therefore, be used to enrich for germline-edited events by merely germinating seeds at high temperature. Germination thermotolerance due to inactivation of NCED4 provides a useful whole-plant selectable phenotype of pleiotropic effects on growth or stress tolerance ( Bertier et al, 2018 ). The Non-Expressor of Pathogenesis-Related PR3 gene from cocoa ( TcNPR3 ) is a suppressor of defense responses and editing it in leaves of cocoa confers an increased resistance to infection with the cacao pathogen Phytophthora tropicalis and elevated expression of downstream defense genes ( Fister et al, 2018 ).…”

Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy

“…Albino calli and shoots were generated in both species. This appears to be the first demonstration of CRISPR/Cas9 in a naturally formed neopolyploid system, and only the third case in species of Asteraceae following Lactuca sativa (Bertier, Ron, Huo, Bradford, & Michelmore, ; Woo et al, ) and Chrysanthemum morifolium (Kishi‐Kaboshi, Aida, & Sasaki, ). This work illustrates the potential of applying CRISPR/Cas9 technology in studies of evolutionary and functional biology and provides a guide for others working on nongenetic model systems.…”

A Revolution toward Gene-Editing Technology and Its Application to Crop Improvement

“…The CRISPR/Cas9 technique was used mainly as a proof of concept in many vegetable crops, e.g., in cabbage, Chinese kale, and watermelon, to induce mutations in the PDS gene [113,114]. In the case of vegetables, CRISPR/Cas9 studies have been performed most frequently in tomatoes, because of the economic value of the crop or the ease of genetic transformation using agrobacterium.…”